HPLC Method of Analysis for Determination and Standardization of Luteolin and Vanillic Acid in Dry Extract of Paronychia Argentea Lam.

Introduction

Paronychia argentea Lam. (Locally known as Rijl El Hamameh) is a perennial herb, distributed widely throughout in Jordan.¹ Several studies showed that Paronychia argentea has hypoglycemic activity,^2-4 and it has been proved to be useful as gastric analgesic, bladder, prostate, abdominal ailments treatment, and stomach ulcers treatment.⁵ It also showed significant alpha amylase⁶ and acetyl cholinesterase (AChE) enzyme inhibitory activity,⁷ the plant extract of Paronychia argentea showed antioxidant activity.⁸ Other in vivo and in vitro studies on different extracts from Paronychia argentea revealed the immunomodulating activity of the plant.⁹ Many chemical compounds have been identified and isolated from Paronychia argentea as: jaceosidin, tricin, nepetin, octadecanoic acid, 1- docosanol, glycerol octadecanoate monoester, β- sitosteryl glucoside, β-sitosterol, and luteolin-3- methyl ether, ethyl-α- D- galacto-pyranoside, D-pinitol(Pinitol is cyclitol) and vanillic acid, luteolin.¹⁰ Vanillic acid and luteolin are considered as the major’s active constituent of Paronychia argentea responsible for its biological activity.¹¹

Scheme 1 Click here to View scheme

 

Material and Methods

Materials

Plant material of P. argentea, aerial part, was collected on April 2016 from Ajloun area, Jordan. The dried plant material (600g) was grounded and soaked in ethanol (90%) for three weeks with frequent agitation, the alcohol solution was and then filtered and evaporated using Rota Vapor to obtain a solid residue (48 g).

Standard luteolin Primary Reference Standard, batch number HWI01784 was purchased from HWI Pharma services GmbH – Germany. Standard vanillic acid Primary Reference Standard, lot number STBD6012Vwas purchased from Sigma Aldrich – Germany, Distilled water, Acetonitrile HPLC grade, Methanol HPLC grade, Acetic acid % HPLC grade 99%.

Methods

High Pressure Liquid Chromatographic Analysis

Preparation of Mobile phase

Distilled water, methanol and acetic acid were mixed at a ratio of (700:300:10) ml respectively then passed through a nylon membrane filter having a pore size of 0.45µm and sonicate to degas.

Preparation of Reference Standards

3.0 mg of Luteolin RS were accurately weighed and transferred to a 5 ml volumetric flask then 3 ml of mobile phase were added and sonicate for 10 minutes. Mobile phase was added to complete the volume to 5ml (Final Concentration 0.60 mg/ml). In a similar way vanillic acid RS solution using was prepared.

Accurately weighed a 20 mg of Vanillic acid RS and transferred to a 50-ml volumetric flask then 30 ml of mobile phase was added, and the solution was sonicated for 10 minutes and made up to volume with the mobile phase(Final Concentration 0.4 mg/ml).

Chromatographic Condition

All analyses were performed using an HPLC HITACHI Chromaster (5160 HPLC pump) & (5410, dual λ   UV absorbance detector), HPLC column ODS-3 (150X4.6) mm, (5µm) and 5310 column oven and auto sampler 5260 were used during the HPLC analysis.

Standard solutions were injected using equal volumes of 20ml of at a flow rate of 1.0ml\minute and a wavelength of 260 nm.

To calculate the percentage of both vanillic acid and luteolin in the sample taken of the dry extract, the following formula was used:

Where

AUC _sample : area of sample.

AUC_STD : area of standard.

C_STD : Concentration standard taken (µg/ml).

P : Potency of Luteolin standard (91.82%)

P : Potency of Vanillic acid (97%)

C _sample : Concentration of Sample taken (µg/ml).

Sample Preparation for HPLC Analysis

4.0 g of dry extract were transferred to a 25 ml volumetric flask, and 15 ml diluent (mobile phase) was added and sonicated for 20 minutes. The volume was completed with diluent (mobile phase) and filtered through 0.45 µm Nylon filter.

Method Validation

The analytical method used for the quantification of Luteolin and Vanillic acid in the P. argentea extracts was validated for linearity, LOD and LOQ, precision, as previously described.

Results and Discussion

Optimization of Chromatographic Conditions

The HPLC conditions were optimized for the mobile phase composition, column temperature, wavelength, and flow rate (Table below). Detection wavelengths were set according to the ultraviolet (UV) absorption maxima of the compounds (260 nm).

Chromatographic conditions
Column	ODS-3 (150X4.6) mm, 5 µm
Column Oven	30°C
Flow rate	1.0 ML/min.
Injection volume	20 µl
Wavelength	260 nm
Flushing solution	Methanol and distilled water at a ratio of 1:1
Runtime	15 minutes

 

Peak of Luteolin and Vanillic acid standard preparations appear at retention time of 1.3 and 5.7 min respectively as shown in figure 5 and figure 6 respectively, and a sample preparation was injected into the chromatograph and two peaks appear at retention times of 1.6 min, 5.7 min respectively as shown in figure 7.

Determination of Major Components in Paronychia Argentea Lam Dry Extract

It was found that the Luteolin content is 0.4% while vanillic acid content is 0.1%

Method Validation

Linearity

The linearity of the assay method of vanillic acid was determined in the range from 25, 40, 50,60,75, 85 and100%, proportional to the concentration relative to the prescribed standard concentration 0.4 mg/ml and the curve was linear over a this large number of concentration and exhibited a linear regression (R² = 0.99981) and slope of 201133.9864.

Statistical analysis are shown in table1 and a plot of area under the curve versus concentration can be seen in Fig. 1.The standard curve was plotted and evaluated for linearity.

The obtained equation for the standard curves was: Equation: Y = AX – B, Where B is the intercept with Y-axis and A is the slope.

Figure 1: Regression for concentrations vs. area of assay vanillic acid standard. Click here to View figure

 

Table 1: Standard curve of assay vanillic acid standard. (Nominal concentration= 0.4 mg/ml).

Injection(µL)	Concentration (µg/ml)	Area	Average	RSD
100	199.0000	39927850	39775006	0.54
		39622161
85	174.1250	34480770	34505485	0.10
		34530200
75	149.2500	29111735	29310849	0.96
		29509963
60	124.3750	24543949	24472215	0.41
		24400481
50	99.5000	19482803	19495800	0.09
		19508796
40	74.6250	14549070	14537364	0.11
		14525657
25	49.7500	9651494	9662163	0.16
		96722831
R2 = 0.99981		Slope= 201133.9864
Intercept = -479056.6964

 

Table 2: Standard curve of assay of Luteolin. (Nominal concentration= 0.6 mg/ml).

Injection(µL)	Concentration(µg/ml)	Area	Average	RSD
20	572.9568	129915	129473	0.48
		129030
17.5	501.3372	105752	105666	0.12
		105580
15	429.7176	88067	87943	0.20
		87818
12.5	358.0980	69181	69222	0.08
		69262
10	286.4784	49206	49530	0.92
		49853
7.5	214.8588	30157	30336	0.83
		30514
5	143.2392	10214	10224	0.14
		10234
R2=	0.99908	Slope=	272.6805
Intercept=	-28733.2679

 

Figure 2: Regression for concentrations vs. area of assay Luteolin standard. Click here to View figure

 

Precision

System Repeatability

In this test, system repeatability tests were examined and standard solutions containing Vanillic acid were Prepared, and injected 10 times into the HPLC system. Average of peak areas and % RSD values were calculated and shows to be within acceptable limits of 0.98%. The resulting areas and RSD values shown in Table 3.

Table 3: System precision for vanillic acid standard.

Area under the curve for 10 replicate injections
Injection #	Standard area of Vanillic Acid
1	110246472
2	111954152
3	109265151
4	110448955
5	112542944
6	111111762
7	110796209
8	108973254
9	111229759
10	110073105
Average	110664176.3
RSD	0.98

 

Analysis Repeatability

For the determination of the repeatability of the extract, six samples obtained from multiple sampling of extract were analyzed, in a single laboratory on a single day. Assay percentage, and RSD values were calculated. The results obtained are listed in Table 4.

Table 4: Analysis repeatability extract.

Dry Extract	Sample #	Assay% of Vanillic Acid	Assay % of Luteolin
	1	0.13	0.43
	2	0.13	0.42
	3	0.14	0.47
	4	0.13	0.42
	5	0.14	0.46
	6	0.13	0.41
	Average	0.13	0.44
	RSD %	3.5	5.1

 

Range

Range is the concentration interval between the upper and lower concentration of analyte for which is shown that the method has suitable level of linearity. Range for determination is extended from 25% to 100% vanillic acid.

Limit of Detection

Limit of detection (LOD) is the lowest concentration of analyte in a sample, which can be detected, but not necessary quantified, under the stated experiment conditions.

For vanillic acid it is estimated by extrapolation of regression line through Y axis from standard curve at low concentration as in the following expression:

The LOD was found to be 0.40 (µg/ml) for vanillic acid which is equivalent to 0.2%   and 1.2(µg/ml) which is equivalent to 0.6% of nominal value of Luteolin respectively.

Limit of Quantitation

The limit of quantitation (LOQ) is the lowest concentration of analyte in a sample, which can be determined with an acceptable accuracy and precision detected.

For Vanillic acid is estimated by extrapolation of regression line through Y axis from standard curve at low concentration as in the following expression (Fig. 3 and 4).

The LOQ was found to be 1.2 (µg/ml) for vanillic acid which is equivalent to 0.6% of nominal value and 9.7(µg/ml) which is equivalent to 4.8% of nominal value of Luteolin respectively (Tables 6, 7 and 8).

Table 5: Standard solution of vanillic acid prepared for calculation of LOD and LOQ.

Injection(µL)	Concentration (µg/ml)	Area	Average	RSD
10	99.5000	19482803	19495800	0.09
		19508796
7.5	74.6250	14549070	14537364	0.11
		14525657
5.0	49.7500	9651494	9662163	0.16
		9672831
R2= 0.99998		Slope= 197661.0452
Intercept = -185347.0000		SD of Intercept = 24027.8748

 

Figure 3: Regression for concentrations vs. area of vanillic acid standard for LOD and LOQ. Click here to View figure

 

Table 6: LOD and LOQ of vanillic acid estimation results.

Parameter	Concentration	Percentage from Nominal
LOD	0.40 (µg/ml)	0.2%
LOQ	1.2 (µg/ml)	0.6%

 

Table 7: Standard solution of luteolin prepared for calculation of LOD and LOQ.

Linearity of Luteolin for LOD & LOQ
Injection (µL)	Concentration  (µg/ml)	Area	Average	RSD
10	286.4784	49206	49530	0.92
		49853
7.5	214.8588	30157	30336	0.83
		30514
5	143.2392	10214	10224	0.14
		10234
R2=0.99982		Slope= 274.4046
Intercept =-28928.5833		SD of intercept = 264.8594
LOD Conc.= 3.19		LOD %= 1.6
LOQ Conc.=9.7		LOQ%=4.8

 

Figure 4: Regression for concentrations vs. area of assay vanillic acid Luteolin standard. Click here to View figure

 

Table 8: LOD and LOQ of Luteolin estimation results.

Parameter	Concentration	Percentage from Nominal
LOD	3.19 (µg/ml)	1.6%
LOQ	9.7 (µg/ml)	4.8%

 

Filter compatibility

Variation of the filter type is described below and the results are reported in Table 9.

Table 9: Filter compatibility results.

Vanillic acid
Filter type	Area of Standard	Change % from unfiltered
Unfiltered	110448955	–
0.45µm Glass	112401742	101.8
0.45 µm Nylon	111021145	100.5
0.45µm PTFE	112251423	101.6

 

Several attempts were used to develop a reverse phase HPLC method of analysis for the separation of the two main components luteolin and vanillic acid available in dry extract of Paronychia.

A suitable mobile phase was established to separate the two major compounds of the extract in one run with a suitable and reasonable time, where both vanillic acid and luteolin were detected at a wavelength of 260 nm, and a sample preparation was injected into the chromatograph and two peaks appear at retention times of 1.3 min, 5.7 min respectively and a runtime of 15 minutes and at a flow rate of 1.0 ml\min using ODS-3 (150X4.6) mm, 5 µm.

A baseline resolution was obtained under the testing of the analysis conditions, as well as the chromatograms of the standards and samples preparation are shown in Fig. ( 5, 6 and 7). this method has been validated as per ICH guidelines for linearity, precision, limit of detection and limit of quantitation for both main active components, and a linear relationship was noticed for the two compounds and the correlation coefficient was R² = 0.9998. With such results of the precision, repeatability LOD, LOQ and filter compatibility, it can be concluded that the method developed and used is precise, sensitive accurate and reproducible, and facilitate quantitative determination of this extract and will accomplish the objective of this study and consequently utilizing a local Jordanian plant since the active materials in the extract are variable. The concentration of luteolin and vanillic acid in the dry extract was 0.4 and 0.1% respectively.

Figure 5: Represent the chromatogram of Luteolin Reference Standard. Click here to View figure

 

Figure 6: Represent the chromatogram of vanillic acid Reference Standard. Click here to View figure

 

Figure 7: Represent the chromatogram of Luteolin and Vanillic Acid in dry extract sample. Click here to View figure

 

Table 10: Rf values, areas under the curve and percentage of the area for Luteolin and Vanillic acid.

Peak Number	Ingredient name	Rf	Area	Theoretical plates	Asymmetry	Area%
1	Luteolin	1.3	33798868	1903	1.2	79.0
2	Vanillic acid	5.7	8971344	511	3.1	21.0

 

Conclusion

The two major peaks of Luteolin and Vanillic acid were simultaneously separated and determined successfully  from the other components in the Paronychia argentea Lam dry extract collected form the plant located in Jordan and was found that the Luteolin content is 0.4% while vanillic acid content is 0.1% using a validated chromatographic method of analysis. These  two compounds  are  considered as a standardized reference of the plant material.

Conflict of Interest

There is no conflict of interest.

Acknowledgments

The Authors acknowledged the technical support of Cargil and its team during analysis of Paronychia argentea Lam in the dry extract and Dr. Isra Dmour for her assistance, and wishes to thank the Deanship of Scientific Research of Al-Ahliyya Amman University. Amman – Jordan for Financial support.

References

1. Al Rawashdeh, I.and Al Rawashdeh, N. Biology Agriculture healthcare, 2016,6, 128.
2. Al-Khalil, S.. International Journal of Pharmacognosy, 1995,33, 317.
   CrossRef
3. Ahmed, S.: Mallick I. and Hasan, M. J. Pharmacogn. Phytochem., 2017, 6: 1780-1787.
4. Afifi, F.: Al-Khalidi, B. and Khalil, E. J Ethnopharmacol. 2005,100(3):314-8.
   CrossRef
5. Hamdan, I. and  Afifi F. J Ethnopharmacol. 2004, 93(1):117-21.
   CrossRef
6. Ali-Shtayeh M.; Yaniv, Z. and  Mahajna J. J Ethnopharmacol. 2000,73(1-2):221-32.
   CrossRef
7. Ferreira, A.; Proença, C.; Serralheiro, M.; Araújo, M. J Ethnopharmacol. 2006, 108(1):31-7.
   CrossRef
8. Tawaha, K.; Alali, F. Q.; Gharaibeh, M.; Mohammad, M.; El-Elimat, T. Food Chemistry, 2007; 104, 1372.
   CrossRef
9. Szallasi, A. Drug Aging., 2001, 18, 561.
   CrossRef
10. Baqi, Y. N. H., University of Jordan. MSc Thesis, 2003.
11. Vriens, J.; Appendino, G.; Nilius, B.Molecular Pharmacology, 2009; 75, 1262.
    CrossRef

---

This work is licensed under a Creative Commons Attribution 4.0 International License.